Expression of Candidate Genes in Zebrafish Blood Islands
Whole-embryo in situ hybridization was performed on embryos at 24 hpf.
(A) slc4a1 was used as control to delineate the intermediate cell mass (ICM, indicated by arrows).
(B) Cloche (clo) embryos were used to show specificity of the hybridizations.
(C–H) Candidates identified in this study.

Morpholino Knockdown of Candidate Genes in Zebrafish Results in Profound Anemia
WT zebrafish embryos were injected at the one-cell stage with the respective morpholinos and stained at 48 hpf with o-dianisidine to detect hemoglobinized cells.
(A) Uninjected (WT) embryos show normal hemoglobinization as indicated by dark brown staining on the yolk sac (arrow).
(B–G) Morpholino-injected embryos. Accurate morpholino gene targeting was verified by RT-PCR (slc25a39, slc22a4, slc22a5, tmem14c, and c1orf69) or real-time quantitative RT-PCR (isca1) on cDNA from uninjected (WT) or morpholino-injected (mo) embryos. β-actin (actb) was used as a control for off-target effects in the RT-PCR. For RT-PCR, (ctrl) indicates no cDNA template control. For real-time quantitative RT-PCR, (ctrl mo) indicates embryos injected with a standard control morpholino.

Mouse Slc25a39 Is Expressed in Hematopoietic Tissues and Is Important for Heme Biosynthesis
(A) Mouse tissue northern blot analysis of Slc25a39 expression.
(B) Mouse Slc25a39 transcripts are localized to blood islands of the yolk sac at early somite stages (E8.5, arrows).
(C and D) Slc25a39 transcripts accumulate most abundantly in the liver, where expression is heterogeneous (E12.5).
(E and F) MEL cells were differentiated for 2 days in media containing 1.5% DMSO prior to (E) transient transfection with myc-tagged Slc25a39 or (F) silencing of Slc25a39 (si-Slc25a39) and/or Slc25a37 (si-Slc25a37) using siRNA oligos. Assays were performed after 2 additional days of differentiation. si-NS indicates silencing using nonspecific control oligos.
(E) Representative western blot using anti-myc (α-myc-Slc25a39) and anti-Slc25a37 antibodies. Equal loading was verified by anti-tubulin.
(F) MEL cells were metabolically labeled with ⁵⁹Fe conjugated to transferrin, and total mitochondrial iron (⁵⁹Fe-Mito) and iron in heme (⁵⁹Fe-Heme) were determined. Results shown are from two independent experiments assayed in duplicate; error bars denote standard deviation.
(G–I) Representative photos of (G) an uninjected, wild-type control zebrafish embryo, (H) a zebrafish embryo injected with a ferrochelatase-specific morpholino, or (I) a zebrafish embryo injected with a slc25a39-specific morpholino. Arrow indicates porphyric red blood cells in circulation.